Brucella melitensis (Category B) is an intracellular bacterial pathogen that causes a debilitating acute disease and establishes a chronic undulating febrile disease in man. Current vaccine strains are virulent in humans which makes them unsuitable for human use. Biological terrorism and the potential use of B. melitensis for biological warfare clearly warrants direct measures for prevention of human brucellosis. The immediate goal of the proposed studies is to generate and classify mutations in these genes according to their effect on survival and persistence in the mouse model. The long-range goal of this project is to expand the fundamental knowledge base for improved disease prevention through safer, more effective vaccines for human brucellosis. Signature-tagged transposon mutagenesis and the TRASH system (transposon site hybridization) based on the mariner transposon will be used to mutate Brucella to identify microbial genes and mechanisms responsible for in vivo survival. Identification of specific virulence genes will be used to derive protective attenuated candidate vaccine strains in mice. Based on the safety and protective immunity of the mouse model, the overall goal is to perform vaccination/challenge experiments using Macaca mulatta non-human primates to predict the safety and efficacy of these strains for human use. Our specific aims are to: 1) Identify B. melitensis genes necessary for survival and virulence using the TraSH system for generating and screening mutants; 2) Determine the safety and protection induced by B. melitensis vaccine candidates in the mouse model; 3) Evaluate safety, protection and host gene expression in response to candidate live B. melitensis vaccines in non-human primates. The central hypothesis is that the genes that compromise survival of B. melitensis are candidates for inactivation in human vaccine strains. Organisms defective in the gene products necessary for intracellular persistence are expected to retain immune stimulation, but should be unable to persist within the host.